Oxidative stress biomarkers of oral and systemic diseases can be found in saliva. However, there is no uniformity for the saliva collection time in these kinds of analyses and saliva composition may change because of mechanical stimulation. Therefore, the aim of this work was to determine the effect of mechanical stimulation for 10 min on the concentrations of vitamin C, vitamin E, total antioxidants and total oxidants in saliva. Saliva samples from individuals of both sexes, aged between 18 and 38 years, were collected for 10 min at 2 minintervals. Saliva flow rate in each 2 min period was measured, as well the total oxidant state, the total antioxidant capacity, vitamin C and vitamin E concentrations. All analyses were performed in triplicate and were determined using colorimetric tests. The results were analysed using t-test, Friedman's test and analysis of variance (ANOVA) for repeated measures. Mauchly's sphericity test was applied and, if necessary, technical corrections were made using the Greenhouse-Geisser test. We found no significant difference between the amounts of saliva produced across the collection times. Total oxidant status, total antioxidant capacity, vitamin C and vitamin E concentrations remained stable. Based on our findings, saliva can be collected for 10 min or less with masticatory stimulation without any variations in the concentration of the variables analysed. However, we suggest using saliva samples after two minutes of mechanical stimulation.